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The Nerve Channel

Nancy Marie Brown

December 1, 2004

The Nerve Channel

The news of science usually concerns what results a scientist has achieved, not how he [or she] has found them. . . . Yet, scientific progress continues to depend on the invention and refinement of tools and techniques. —Joshua Lederberg (1986)

"Just take a pair of goggles and go in," chemist Andrew Ewing said, as I stood before graduate student Jody Mesaros's lab.

Among the layers of notes, cartoons, and signs papering the door were two that stood out: a yellow "Warning: Radioactivity" and a hand-lettered "The Treehouse." Two pairs of green plastic goggles hung from tacks. I put one on as directed, felt immediately foolish (like a fish in a bowl), hesitated again, and went in.

The small room was divided into two narrow corridors by a high bank of equipment. Mesaros, looking equally goofy in her goggles, was in the near run, rapidly scooping crushed ice into an aluminum-foil bread pan. She looked mildly alarmed to see me, although she'd invited me the day before to watch the experiment that had won first prize for physical sciences at Penn State's 1994 Graduate Research Exhibition.

"It's not working well today," she said, tamping the ice with her thumb. "I'm getting a lot of condensation on the top plate—it's really humid in here—I can't see a thing." She dried a hand, stretched it out in greeting.

In the aluminum bread pan, surrounded by ice and nearly buried under gobs of petroleum jelly, was a small, flat, glassy plate—quartz, actually—roughly the size of a matchbox. Another plate, invisible, was beneath it, with a rim of tiny beads glued between to make a channel.

To one end of this quartz sandwich were fixed two yellow fiber-optic bundles, one of which headed out toward a laser, the other into a computer.

Over the opposite end jutted a thin strip of wood, rather like a tongue depressor, which could be moved back and forth along the beveled edge of the plates with a motor. Stuck to the stick's underside was what looked like a long hair, golden, poking at the gap between the plates, its tail end swirling up and around and finally down into a thimble-sized vial of liquid some distance away. This "hair" was a fused silica capillary, its hollow central bore too small to see.

According to Ewing, the system was capable of separating and identifying picoliter quantities of chemicals—a quantity one scientist has compared to a billionth of a water drop, the amount, say, released by a single, excited nerve cell in a snail.

"I wanted to do it cheap," Mesaros said, acknowledging the aluminum pan and tongue-depressor-like stick that held the capillary in place. "We didn't know if it was going to work."

She smiled, agreeing that the set-up didn't look like much. She is amused, she added, when outside scientists come to see it: They glance, nod, clear their throats, and say, "Well, let's have a look at those snails."

The snails in question are Planorbis corneus, the Ram's Horn Snail, a large species of the common pond-dwelling, plant-eating trumpet snail, with a brown spiral shell about an inch across. To look at, they're hardly a bundle of nerves.

Yet their nerves are what attracted Ewing. They are large, close to the surface. Perfect for the delicate prying and sampling of his hair-thin tools.

"Take an electrode. Put it on top of a nerve cell. There's no response." Ewing's done this, fixing a tiny electrical fiber inside a slightly less tiny capillary tube and situating it a few microns outside of a cell. "But if the cell is stimulated"—with nicotine, for example, or an amphetamine—"you get a bunch of spikes." Standing, he traced over a spiky graph, once erased, on the blackboard beside his desk, a record of the neurotransmitters the electrode detected. "Each spike is due to one vesicle."

A nerve cell, or neuron, consists of a cell body, with its antennae-like dendrites to receive incoming signals, and a long stalk of an axon, from the fingery tip of which new signals are sent across the synapse—the gap—to another neuron. The axon's tip contains the vesicles—sacs—filled with neurotransmitters, the chemical signals to be released (or as Ewing said, "dumped") when the cell is stimulated.

There are some 28 billion neurons in the body, 10,000 million in the brain alone. They vary widely in body size and axon length (up to a meter long), and in the chemical and physical mechanisms they use to communicate. Such variety in the cells means that measuring them in the aggregate can obscure their true nature: It calls for analyzing them one-by-one.

For such unicellular probing are Ewing's capillary-based chemical separation techniques designed: Recently, for example, members of his research team have been able to measure the levels of the neurotransmitters dopamine and serotonin in the cell bodies of individual Planorbis nerves, confirming that nerve axons hold two different kinds of vesicles (the one deeper inside the cell they suggest is for "longer term storage"). They have also learned that the amount of neurotransmitter held in a vesicle can be changed by drugs like amphetamines.

This cleverness with instrumentation has earned, Ewing, at 37, the 1994 Penn State Faculty Scholar Medal in the physical sciences. According to his nomination for the honor, Ewing has singlehandedly developed a new area of bioanalysis at the level of single nerve cells. . . . [His] contributions to the area of single cell analysis are changing the way that we approach problems in neurobiology.

"I would like to know more," Ewing said, when I asked him to sum up his lab's overall goals, "about how nerve cells transmit their signal from one cell to another.

"Everyone always thinks of neurotransmission as happening across the synapse," he continued. "But when you stimulate the cell body, you get a massive release of neurotransmitters. Why? What role does this play in our nervous system? Nothing's been developed to look at this."

Another puzzle concerns the fact that vesicles also, according to Ewing, dump ascorbic acid, the energy source ATP, a protein called chromogranin A (which the vesicle needs to bind its parts together), and, most mysteriously, various amino acids bound together as peptides. "No one has a clear idea what peptides do in the neurotransmission process," Ewing said. "We don't have good techniques to look at them."

"The electrode oxidizes everything that's easily oxidized," he said. "It wouldn't detect ATP or chromogranin-A, and it can't distinguish among norepinephrine, epinephrine, and ascorbic acid." It won't pick up peptides, nor will it tell you what was released first, what last.

Mesaros's channel, however, ultimately may.

"The whole point of what we're doing," Mesaros explained, turning back to her ice-scooping, "is this:

"Say you have a normal capillary sticking in a snail cell. You can pull up two seconds'-worth of fluid. If you pull up too much more, you'll never get a separation." With too much material, said Mesaros, a detector trying to separate three molecules, for instance, would produce only "a broad band with three humps on it" rather than three nice crisp peaks; where the signal from one molecule ends and another begins is impossible to tell, "because they are all smeared into each other."

Even if some new technique made it possible to clearly separate the various molecules in a minute-long "plug," as Mesaros called it, the separation still wouldn't reveal what had happened when.

"Maybe the cell fired a whole lot at first," she said, "then nothing. Thirty seconds later, it released again, a small release . . . Who knows what it did during that time period of a minute? You'll just get a broad band of chemical."

As an undergraduate at Temple University, Mesaros had been told to build an instrument to ring a bell if the fluorine level in her tapwater rose above 1 part per million. The experience pushed her toward graduate school in analytical chemistry. "When I visited Andy's lab at Penn State," she recalled, "I really liked the research. Everyone seemed really happy."

Joining the group, she was given a list of experiments in progress and of ones Ewing wanted to begin. "What I really wanted to do was to come in and think of an idea by myself," Mesaros said, "but I wasn't at that stage in my education yet." She decided to take on a project that Ewing and Johan Roeraade of the Royal Institute of Technology in Stockholm had concocted at a conference: using a capillary and a thin channel to take samples of the fluid released by a nerve cell and separate them into their parts continuously, second by second, over the course of several minutes—theoretically, as long as a snail nerve can be kept alive—keeping track of which chemicals were released when, in what quantity.

"It needs a lot of work before people can routinely work on a snail," Mesaros said, explaining how her set-up works.

The liquid in the vial was a mixture of four dansylated amino acids (the dansyl group makes the molecules glow in the laser light), of known proportions. "We know exactly what's in there," she said. "We're just trying to standardize the system for when we have a real sample."

While she spoke, the hair-like capillary was busily sucking up a picoliter at a time of solution and conveying it to the minuscule gap between the quartz plates. There, pulled along by an electric current, the four amino acids went traveling down the rectangular channel, spreading out, as they went, to form four tilted bands (as picked up by the fiber-optic detector and graphed on the computer screen), each band containing molecules of only one amino acid. Reading along the horizontal axis of the graph tells which amino acids came out when.

"Usually you can see the separated molecules in the channel," said Mesaros, pointing to the top quartz plate, now completely fogged over in the humid air of the lab. "They glow yellow. That's one reason I'm glad fluorescence was the first detector we tried. You can see if everything's working.

"Peter's got a harder time of it," she continued, scooping in more ice. She meant Peter Gavin, another Penn State graduate student working for Ewing. Gavin's project, based on Mesaros's success, is to develop a more sophisticated detection scheme than her bundle of fiber optics: Many of the chemicals Ewing wants to sample and separate in Mesaros's channel can't be made to fluoresce. "Peter can't see it, can't tell if it's working," added Mesaros. "You really lose a lot of time that way."

The trick of it all is the motor that moves the capillary (affixed to its tongue-depressor-stick) step by step, 25 microns at a time, from one end of the channel mouth to the other. "If there was no movement," Mesaros explained, "the bands of material would smear into each other. With capillary movement, a very small amount of material is deposited into the channel, and a complete separation is possible." Sampling and separating can thus run continuously—Mesaros has so far clocked seven minutes, running the capillary back and forth past the channel mouth. "Some people like to think of the channel as several hundred capillaries side by side," she added. "The capillary movement allows a small amount of material to be injected into a hypothetical 'capillary' at each 25 micron step."

"What this allows us to do," concluded Ewing, explaining it at the blackboard in his office, "is to look at the dynamics of neurotransmitter release from single vesicles.

"It's a step," he added. "We'll do interesting neuroscience with this alone. Jody's work will be with adrenal cells, in culture.

"But my real goal is to go after the snail."

Jody Mesaros won first prize in the Physical Sciences Division of the 1994 Graduate Research Exhibition; she received her Ph.D. in analytical chemistry in December 1994 from the department of chemistry, the Eberly College of Science, University Park, PA 16802; 814-865-6553.

Her adviser, Andrew G. Ewing, Ph.D., is professor of chemistry; 863-4653. Peter Gavin is a doctoral candidate in analytical chemistry. This work was supported by the NSF, the NIH, and the Office of Naval Research.